To investigate the presence of and the potential biological roles of protein tyrosine phosphorylation in the glioblastoma pathogenesis two-dimensional gel electrophoresis- (2DGE-) based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain Ciproxifan maleate structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress stress response and cell migration. Those data show 2DGE-based Western blotting MS/MS and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma. 1 Introduction Tyrosine phosphorylation that is Ciproxifan maleate an addition of phosphogroup (-HPO3 to -OH or -H3PO4 to -NH2) to the tyrosine residue is a type of protein posttranslational modification that plays key roles in the signal transduction and participates in many physiological and pathological processes such as growth proliferation differentiation aging cancer and inflammatory diseases [1-3]. Tyrosine phosphorylation and dephosphorylation are a reversibly dynamic mechanism that is regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) . Moreover tyrosine kinase phosphorylation generally occurs within a consensus pattern/motif [R/K]-x(2)-[D/E]-x(3)-Y or [R/K]-x(3)-[D/E]-x(2)-Y (Y = the phosphorylation site) [5-7]. Currently 518 human protein kinase genes  including 90 known tyrosine kinases that include 58 receptor tyrosine kinases (RTKs) [9 10 and 107 tyrosine phosphatases  have been discovered for potential targets of anticancer drugs most tyrosine kinases are regulated negatively and only activated under certain conditions  and interestingly tyrosine kinases accounting for 0.3% of genome contribute to a large proportion (30%) of 100 known dominant oncogenes [10 12 Tyrosine phosphorylation (accounting for only ~0.05%) is a low abundance event in the phosphoproteome relative to phosphorylation at the serine (accounting for ~90%) and threonine (accounting for ~10%) residues in eukaryotic cells [1 3 10 13 However characterization of altered modification and functional activities of phosphotyrosine-containing proteins in different types of cancers has helped in the discovery of specific tyrosine kinase inhibitors to treat a cancer [9 14 Thus it emphasizes the scientific importance of investigating phosphotyrosine-containing proteins in a cancer. The most common characteristics of glioblastoma are highly invasive growth and aggressive infiltration into surrounding normal brain which Ciproxifan maleate causes the failure of current therapies to control glioblastoma with a median survival of 9-12 months in spite of the improvement of the current therapies such as surgery radiotherapy and chemotherapy . The molecular mechanisms of glioblastoma remain unclear. It is necessary to discover novel biomarkers for novel therapeutic strategy to control its invasive growth. Many studies have indicated that tyrosine phosphorylation is extensively associated with pathophysiological processes of glioma including angiogenesis [16-21] immune response  and invasive growth and migration [23-27]. Tumor angiogenesis is an important reason why glioblastoma is capable of highly invasive growth and aggressive infiltration. Many positive and negative regulating factors of angiogenesis are involved in the tyrosine phosphorylation [16-21] such as Rabbit Polyclonal to A1BG. vascular endothelial growth factor (VEGF) and its receptor (VEGFR) [16 17 21 28 epidermal growth factor Ciproxifan maleate (EGF) and its receptor (EGFR) [15 19 20 29 platelet-derived growth factor (PDGF) and its receptor (PDGFR) [29 33 leucine-rich repeat C4 (LRRC4)  the uPA/uPAR system  ERK1/2 signaling  and the focal adhesion kinase signaling pathway [36 37 A series of protein kinases associated with glioma are studied including RTK (EGFR ErbB2 ErbB3 IGF-IR and KIT) [30-32 38 Lyn kinase/Src kinase  Akt and focal adhesion kinase [27 36 37 42 43.